NL2037597A - Method for screening lactobacillus casei fermentation agent metabolizing sucrose - Google Patents
Method for screening lactobacillus casei fermentation agent metabolizing sucrose Download PDFInfo
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- NL2037597A NL2037597A NL2037597A NL2037597A NL2037597A NL 2037597 A NL2037597 A NL 2037597A NL 2037597 A NL2037597 A NL 2037597A NL 2037597 A NL2037597 A NL 2037597A NL 2037597 A NL2037597 A NL 2037597A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6818—Sequencing of polypeptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/245—Lactobacillus casei
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
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- Bioinformatics & Computational Biology (AREA)
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Claims (6)
1. Werkwijze voor het screenen van een lactobacillus casei fermen- tant die sucrose metaboliseert, omvattende de volgende stappen: Sl, amplificatie van een sacA-gen: het extraheren van genomisch DNA van de te testen lactobacil- lus casei en het verkrijgen van een volledige sequentie van het sacA-gen van de te testen Jactobacillus casei door amplificatie, waarin PCR-primers die het sacA-gen amplificeren nucleotidesequenties bevatten die zijn weergegeven als SEQ ID NO.1 en SEQ ID NO.2; S2, bepaling van een aminozuursequentie van sucrose-6- fosfohydrolase van de te testen lactobacillus casei: op basis van de volledige sequentie van het sacA-gen verkregen in S1, bepaling van de aminozuursequentie van het sucrose-6- fosfohydrolase van de te testen lactobacillus casei met behulp van software; en S3, vergelijking van de aminozuursequentie van het sucrose-6- fosfohydrolase vergelijking van de aminozuursequentie, die wordt weergegeven als SEQ ID NO.3, van het sucrose-6-fosfohydrolase van de te testen lactobacillus casei, verkregen in S2, en vaststelling dat de te testen lactobacillus casei in staat is sucrose te metaboliseren.
2. Werkwijze volgens conclusie 1, verder omvattende de volgende stappen: S4, controle van het gebruik van de te testen lactobacillus casei op sucrose: S41, activeren van de te testen lactobacillus casei; en S42, bepalen van het gebruik van de te testen lactobacillus casei op sacharose.
3. Werkwijze volgens conclusie 1, waarbij het extraheren van het genomisch DNA van de te testen lactobacillus casei in S1 specifiek de volgende stappen omvat: enten van de te testen lactobacillus casei in een kweekmedium,
kweken van dezelfde op een stationaire manier, wanneer OD600 = 0,8-1. 0, bacteriële vloeistof nemen om te centrifugeren, het su- pernatant verwijderen, een TES-oplossing aan het neerslag toevoe- gen om de bacteriën te resuspenderen, centrifugeren, het super- natant verwijderen, vervolgens lysozym aan de bacteriën toevoegen om de bacteriën te resuspenderen, digestie uitvoeren, vervolgens RNase aan wellmix toevoegen, vervolgens een protease K-oplossing aan wellmix toevoegen, een bufferoplossing GB toevoegen voor os- cillatie, behandeling in een waterbad uitvoeren, watervrije etha- nol aan wellmix toevoegen, centrifugeren en afvalvloeistof afgie- ten; een bufferoplossing GD toevoegen, centrifugeren en de afval- vloeistof afgieten; een wasoplossing PW toevoegen om te wassen, centrifugeren en de resterende wasoplossing in een adsorberend ma- teriaal drogen; en een voorverwarmde TE-bufferoplossing druppels- gewijs toevoegen, centrifugeren, monsters verzamelen en de mon- sters bij een lage temperatuur bewaren voor later gebruik.
4. Werkwijze volgens conclusie 1, waarbij in S1 het sacA-gen wordt geamplificeerd met behulp van een PCR-technologie; een Ex Tag HS- enzym wordt geselecteerd om een 25 pL systeem te maken; en de PCR- reactieomstandigheden zijn: 95°C, 3min; [95°C, 30 s; 60°C, 30s; en 72°C, 2 min] x 30 cycli; 72°C, 5 min; en 4°C.
5. Werkwijze volgens conclusie 2, waarbij in 54, S41 het activeren van de te testen lactobacillus casei de volgende specifieke stap- pen omvat: het enten van de te testen lactobacillus casei in een MRS vloeibaar kweekmedium, en het kweken van de te testen lactobacil- Jus casei bij 37+1°C gedurende 12 uur, en het uitvoeren van stamac- tivering.
6. Werkwijze volgens conclusie 2, waarbij in S4, S42 het bepalen van het gebruik van de te testen lactobacillus casei op de sucrose de volgende specifieke stappen omvat: eerst bepalen of de te testen lactobacillus casei in staat is om de sucrose te benutten; de te testen lactobacillus casei inocu-
leren in een vloeibaar MRS-kweekmedium en de te testen lactobacil- Ius casei gedurende 12 uur bij 37+1°C kweken en het sucrosemetabo- lisme door de stam detecteren; vervolgens de stam waarvan is vastgesteld dat deze de sacharo- se kan gebruiken, gebruiken als basis voor gefermenteerde melk van een fermentatiemiddel en de sacharosegebruiksgraad berekenen; bereiding van een gefermenteerde melk: rauwe melk en de sacha- rose mengen, homogeniseren en pasteuriseren, en na koeling een fermentatiegrondstof bereiden; en de te testen geactiveerde lacto- bacillus casei in de fermentatiegrondstof enten met een toevoeging van 5x10° CFU/g, en een kweek bij 37 °C uitvoeren om de gefermen- teerde melk te verkrijgen; en detectie van het sacharosegehalte: nauwkeurig afwegen van een sacharosestandaard in een maatkolf van 100 ml, aanvullen tot 100 ml met ultrazuiver water, waarin de massaconcentraties van de sa- charose respectievelijk 3, 2,5, 2, 1,75, 1,50, 1,25, 1,0, 0,75, 0,50 en 0,25 mg/mL zijn; plotten van een sacharosestandaard in een maatkolf van 100 ml. 25 mg/mL; een standaardcurve uitzetten door de concentratie van de standaard als horizontale coördinaat en een piekoppervlak als longitudinale coördinaat te gebruiken; de gefer- menteerde melk in een bekerglas wegen en gedeïoniseerd water toe- voegen om het geheel op te lossen met een magneetroerder; achter- eenvolgens een Carrez-reagens A en een Carrez-reagens B toevoegen en na volledig oplossen een mengsel verkrijgen; het mengsel cen- trifugeren en het supernatans nemen; het supernatans verdunnen met gedestilleerd water en filtreren met behulp van een filtermem- braan; in combinatie met een chromatografische kolom en een diffe- rentiële brekingsindexdetector (RID), waarbij een zwavelzuuroplos- sing als mobiele fase wordt gebruikt en het sacharosegehalte in een gefermenteerd melkmonster wordt gedetecteerd met behulp van een externe standaard werkwijze, zodat de sacharosebenuttingsgraad kan worden berekend.
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